Internexin Causes Abnormal Neurofilamentous Accumulations

The Journal of Neuroscience, April 15, 1999, 19(8):2974–2986

Overexpression of ␣-Internexin Causes Abnormal Neuroﬁlamentous Accumulations and Motor Coordination Deﬁcits in Transgenic Mice

Gee Y. Ching,1 Chung-Liang Chien,2 Roberto Flores,1 and Ronald K. H. Liem1 1Departments of Pathology and Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032, and 2Department of Anatomy, National Taiwan University School of Medicine, Taipei, Taiwan 100, Republic of China

␣-Internexin is the first neuronal intermediate filament (IF) pro- Behavioral tests demonstrate that these mice have a deficit in tein expressed in postmitotic neurons of the developing ner- motor coordination as early as 3 months of age, consistent with vous system. In the adult, its expression is restricted to mature the morphological neuronal changes. Our data further demon- neurons in the CNS. To study the potential role of ␣-internexin strate that the neurofilamentous inclusions also lead to progres- in neurodegeneration, we have generated transgenic mice that sive loss of neurons in the aged transgenic mice. The motor overexpress rat ␣-internexin. The total levels of ␣-internexin coordination deficit and the loss of neurons are transgene expressed in the hemizygous and homozygous transgenic mice dosage-dependent. These data yield direct evidence that high were ϳ2 and ϳ3 times the normal level, respectively. Overex- levels of misaccumulated neuronal IFs lead to neuronal dys- pression of ␣-internexin resulted in the formation of cerebellar function, progressive neurodegeneration, and ultimate loss of torpedoes as early as 1 month of age. These torpedoes are neurons. Moreover, the degrees of neuronal dysfunction and abnormal swellings of Purkinje cell axons that are usually seen degeneration are proportional to the levels of misaccumulated in neurodegenerative diseases involving the cerebellum. EM neuronal IFs. studies showed accumulations of high levels of IFs and abnormal organelles in the torpedoes and soma of Purkinje cells, as Key words: ␣-internexin; neurofilament; intermediate fila- well as in the large pyramidal neurons of the neocortex and in ment; neurodegeneration; cytoskeleton; transgenic mice; motor the ventral anterior and posteromedial nuclei of the thalamus. deficits

␣-Internexin is a neuronal intermediate filament (IF) protein that son’s disease (Munoz et al., 1988; Sasaki et al., 1989; Galloway et is expressed earlier and more abundantly than the neurofilament al., 1992; Trojanowski and Lee, 1994). Recent transgenic mouse triplet proteins (NFTPs) throughout the developing CNS (for models have shown that overexpression of human NF-H (Cote et review, see Fliegner and Liem, 1991). As development continues, al., 1993) or mouse NF-L (Xu et al., 1993) results in axonal the levels of ␣-internexin decrease, whereas those of the NFTPs swellings and perikaryal accumulation of neuronal IFs in motor increase (Kaplan et al., 1990; Fliegner et al., 1994). In adult CNS, neurons, as well as skeletal muscle atrophy. The mice develop ␣-internexin is colocalized with the NFTPs in most axons, but is signs of muscle weakness and reduced kinetic activity, reminis- present at lower levels in large neurons and is found in cerebellar cent of those found in ALS. Transgenic mice expressing a point granule cells, which lack the NFTPs (Kaplan et al., 1990). mutant of NF-L show neuronal abnormalities resulting in motor ␣-Internexin can self-assemble and coassemble with each of the neuron death and skeletal muscle atrophy (Lee et al., 1994). NFTPs into filaments, whereas the NFTPs are obligate hetero- NF-H mutations have also been found in sporadic ALS patients polymers (Ching and Liem, 1993, 1998; Lee et al., 1993). These (Figlewicz et al., 1994). ␣ properties suggest that -internexin may play a role in stabilizing Overexpression of wild-type or mutant NFTPs does not always small-diameter axons and act as a scaffold on which the NFTPs result in ALS-like phenotypes. Transgenic mice that express a coassemble during development. NF-H/␤-galactosidase fusion protein show perikaryal accumula- Abnormal accumulations of neuronal IFs are pathological hall- tions of IFs in their motor neurons but do not develop motor marks of many neurodegenerative diseases, such as amyotrophic neuron degeneration (Eyer and Peterson, 1994). The animals lateral sclerosis (ALS), Lewy body-type dementias, and Parkin- begin to show tremors and selective Purkinje cell loss only at 1 year of age (Tu et al., 1997). Their Purkinje cells contain Lewy Received Nov. 9, 1998; revised Jan. 28, 1999; accepted Jan. 28, 1999. body-like inclusions. Transgenic mice that express wild-type hu- This work was supported by Grant NS15182 from the National Institutes of man NF-M have normal motor neurons in the spinal cord and Health. C.-L. Chien was supported by Grant NSC 88-2314-B-002-119 from National exhibit perikaryal accumulations of neurofilaments in other neu- Science Council, Taiwan. We thank Dr. Frank Costantini and Ms. Xiao-lin Liang for generation of transgenic mouse founders, Dr. Mary Bach for helpful advice on the rons at 12, but not 3, months of age (Vickers et al., 1994). behavioral experiments, Ms. Beth Rosen and Mr. Reilly Coch for their excellent Surprisingly, transgenic mice that overexpress wild-type mouse technical assistance, Mr. Adam Nguyen and Ms. Allie Lui for assistance in DNA extraction and tissue sectioning, and Drs. Dongming Sun and Conrad Leung for NF-H or NF-M show neither muscle atrophy nor motor neuron their help in photographic printing. loss, despite prominent axonal swellings and perikaryal neurofila- Correspondence should be addressed to Dr. Ronald Liem, Department of Pathol- mentous accumulations in motor neurons (Wong et al., 1995; ogy, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032. Marszalek et al., 1996). Copyright © 1999 Society for Neuroscience 0270-6474/99/192974-13$05.00/0 Thus, overexpressions of different NFTPs have differential Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deficits J. Neurosci., April 15, 1999, 19(8):2974–2986 2975

effects on neurons. Because ␣-internexin differs from the NFTPs precipitated protein obtained after dialysis was solubilized by boiling in assembly properties and expression pattern, its overexpression briefly in 10 mM Tris-HCl, pH 7.5, containing 1% SDS. For immunization, the purified antigen (100 ␮g/immunization) was mixed with 0.2 ml may cause a different type of neuropathy and provide additional saline containing 25 ␮g of monophosphoryl lipid A plus synthetic treha- insights into mechanisms of neuronal dysfunction and neurode- lose dicorynomycolate emulsion (Ribi Immunochem Research), an ad- generation. Our present study shows that subpopulations of CNS juvant, and injected subcutaneously in two sites on Balb/c mice. Booster neurons appear to be relatively more vulnerable to filamentous injections were given every 3 weeks. Two weeks after the last antigen/ misaccumulations induced by ␣-internexin overexpression. More- adjuvant injection, another boost was given without adjuvant. Three days later, spleen cells were removed from the immunized mice for fusion over, the transgenic mice show a transgene dosage-dependent with NS-1 myeloma cells, and monoclonal antibodies were produced deficit in motor coordination. according to the method of Kohler and Milstein (1976). After immunoblotting and immunohistochemical screening, two monoclonal antibod- MATERIALS AND METHODS ies, mAb␣12 and mAb␣3G10, were isolated. mAb␣12 recognizes only rat ␣-internexin, and mAb␣3G10 reacts with both mouse and rat Generation of transgenic mice. To obtain a full-length rat ␣-internexin ␣-internexin. genomic clone, a 6.4 kb BamHI–XhoI fragment and a 14.5 kb XhoI Antibodies. The following antibodies were obtained commercially (Sig- fragment, isolated from the rat ␣-internexin genomic clones ␭␣G-1 and ma, St. Louis, MO): mouse monoclonal antibodies to NF-L, NF-M, and ␭␣G-2 (Ching and Liem, 1991), respectively, were ligated to the BamHI– NF-H (clones NR4, NN18, and N52, respectively); SMI31 and SMI36, SalI digested ␭DashII phage vector (Stratagene, La Jolla, CA). The which detect the phosphorylated epitopes on NF-M and NF-H; SMI32, resulting genomic clone was designated ␭␣D-1. The transgene construct which recognizes the dephosphorylated forms of NF-M and NF-H; and pSP72N-␣ES16K was cloned by ligating the 13 kb EcoRI fragment from tau-2, which detects both phosphorylated and dephosphorylated forms of ␭␣D-1 and the 3.5 kb EcoRI–SalI fragment from ␭␣G-2 together with the tau. Rabbit polyclonal antibody to calbindin 28 kDa was purchased EcoRI–SalI digested pSP72N plasmid vector, which was modified from (Swant, Bellinzona, Switzerland). Rabbit polyclonal antibody to GFAP pSP72 (Promega, Madison, WI) by changing the EcoRV cloning site to a was previously described (Wang et al., 1984). NotI site. To obtain the transgene fragment for generation of transgenic Western blot analysis. M mice, the 16.5 kb NotI–SalI fragment from pSP72N-␣ES16K, consisting Mouse tissues were homogenized in 10 m ␣ Ј Tris-HCl, pH 7.5, and 1% SDS, boiled for 5 min, and then centrifuged at of the entire rat -internexin gene with 1.2 kb of its 5 flanking sequence ϫ and 3.5 kb of its 3Ј flanking sequence, was isolated from agarose gels and 13,000 g for 5 min to remove insoluble materials. The resulting purified by glass powder. The resulting 16.5 kb transgene fragment was supernatant contained total proteins extracted from the tissues. Protein further purified by Sephadex G50 spin columns (Boehringer Mannheim, concentrations were determined by Bradford assay (Bradford, 1976). Equal amounts of proteins were electrophoresed in 8 or 10% Indianapolis, IN) and prepared at 3 ␮g/ml in the 10 mM Tris-HCl, pH polyacrylamide-SDS gels (Laemmli, 1970) and then electrotransferred to 7.4, 10 mM NaCl, and 0.2 mM EDTA buffer. Transgenic mouse founders were generated by microinjecting the transgene fragment into fertilized nitrocellulose filters (Towbin et al., 1979). The filters containing proteins were incubated in PBS containing 5% bovine serum albumin for 30 min, eggs isolated from B6CBA F1 /J mice (The Jackson Laboratory, Bar Harbor, ME) according to the protocol of Hogan et al. (1986). Two washed with PBS, and incubated in PBS containing primary antibodies transgenic mouse lines ␣16K-T5 and ␣16K-T21 were established. Hemi- for 1 hr. After several washes, they were incubated in PBS containing zygous offspring were obtained by breeding the transgenic mice with horseradish peroxidase-conjugated secondary antibodies for 30 min. They were subsequently washed and treated with enhanced chemilumi- B6CBA F1 /J, and homozygous offspring were obtained by breeding the transgenic mice within each line. nescence (ECL) reagents (Amersham, Arlington Heights, IL) for 1 min To determine the presence of the transgene in the mice, small tail and exposed to x-ray films. Several exposures of an autoradiogram were pieces were cut from the mice, and DNA was extracted from these tails used for densitometric analysis. Bam Immunocytochemistry. Mice were anesthetized and perfused with PBS as described (Hogan et al., 1986). Tail DNA was digested with HI, ␮ separated on 0.8% agarose gels, and transferred to Zeta-Probe GT containing 4% paraformaldehyde. Frozen cryostat sections of 10–15 m membranes (Bio-Rad, Hercules, CA). The 4.4 kb BamHI fragment from thickness were prepared from the fixed tissues. The tissue sections were ␭␣G-2, consisting of the third exon and 3Ј flanking sequence of the rat incubated in ice-cold methanol for 10 min, washed, and incubated in PBS ␣-internexin gene, was used as a hybridization probe to detect the rat but containing 5% normal goat serum for 30 min. They were washed with not the mouse ␣-internexin gene. To determine which transgenic mice PBS and then incubated in PBS containing primary antibody for 1 hr. were homozygous with respect to the transgene, equal amounts of tail After several washes, they were incubated in PBS containing horseradish DNA from the hemizygous and homozygous mice were used for South- peroxidase-conjugated secondary antibody for 30 min. They were then ern blot transfer, and a twice stronger rat ␣-internexin hybridization washed and treated with PBS containing 0.012% hydrogen peroxide and ␮ ␮ signal on the autoradiogram indicated the homozygosity of the mice. To 0.5 g/ l diaminobenzidine (DAB). The tissue slides were mounted with check the accuracy of the quantities of tail DNA on Southern blots, the Permount (Fisher Scientific, Houston, TX). The DAB/peroxidase- membranes were rehybridized with a probe for the endogenous cdk5 immunostaining procedure was modified for some primary antibodies: gene (Sun et al., 1996), which served as an internal reference. The methanol incubation was omitted, 0.1% Triton X-100 was added to PBS, homozygosity was further confirmed by breeding the mice. and tissues were incubated with primary antibody overnight at 4°C. Production of mouse monoclonal antibodies. A 0.8 kb BglII–BamHI Indirect immunofluorescence staining using rhodamine- or fluorescent- fragment encoding the C-terminal amino acid residues 340–505 of rat conjugated secondary antibodies was performed as previously described ␣-internexin and consisting of the SV40 poly A signal was isolated from (Ching and Liem, 1993). pRSV-␣ (Ching and Liem, 1993), blunt-ended with Klenow, and at- Electron microscopy. Mice were anesthetized and perfused with 4% tached to BamHI linkers (CGCGGATCCGCG). After BamHI digestion, paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer, pH the resulting 0.8 kb fragment was cloned into the BamHI site on the 7.4. Tissue blocks were immersed in the same fixative for 24 hr at 4°C, pET16b vector (Novagen, Madison, WI). The clone, designated ␣BB, rinsed in cacodylate buffer, and post-fixed in 1% osmium tetroxide for 2 was transformed into the bacterial strain BL21(DE3) for expression. hr. After three washes with cacodylate buffer, each sample was dehy- Expression of the ␣-internexin C-terminal peptide, which also contains drated in a graded series of ethanol and embedded in Epon-Araldite an N-terminal histidine tag encoded by the pET16b vector, was induced resin. Plastic sections (1 ␮m) were stained with toluidine blue and by 0.5 mM isopropyl-␤-D-thiogalactopyranoside for 4 hr. Bacterial cell examined for light microscopy. Ultrathin sections were stained with pellets were suspended in binding buffer (5 mM imidazole, 0.5 mM NaCl, uranyl acetate and lead citrate and examined with a Hitachi H-7100 and 20 mM Tris-HCl, pH 7.9) and sonicated, and the cell lysate was electron microscope. centrifuged at 20,000 ϫ g for 15 min at 4°C. The resulting pellet was Behavioral tests. Hemizygous, homozygous, and nontransgenic mice of resuspended and incubated in binding buffer containing 6 M urea on ice 3 and 6 months of age were tested between 1:00 and 5:00 P.M. in all for 1 hr. The cell extract was centrifuged at 39,000 ϫ g for 20 min at 4°C. behavioral experiments. The genotypes of the mice were unknown to the The supernatant was filtered through a 0.45 ␮m membrane and then observer at the time of the tests. The apparatus were cleaned thoroughly purified by column chromatography using His⅐Bind metal chelation res- after each mouse was tested. Statistical significance in performance ins under denaturing conditions according to the manufacturer’s protocol differences between the genotype groups were calculated using the F test. (Novagen). Protein fractions containing the purified ␣-internexin peptide The open-field test was used to measure the overall locomotor activ- were dialyzed extensively against 10 mM Tris-HCl, pH 7.5, at 4°C. The ities and motor posture patterns (Gerlai et al., 1993). The open-field 2976 J. Neurosci., April 15, 1999, 19(8):2974–2986 Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deficits apparatus was a large opaque cage (12 ϫ 28 cm) whose bottom was rat ␣-internexin was expressed in the brains (with cerebella ϫ covered with a 4 4 cm square grid. Mice were habituated to the removed), cerebella, and spinal cords of the 3-month-old hemi- open-field apparatus twice: 1 hr on the day before testing and 1 hr on the zygous mice from ␣16K-T5 and ␣16K-T21 at approximately the test day. Individual mice were placed in the center of the cage, and their ␣ movements were quantified for 20 min. The following behaviors were same levels as the endogenous mouse -internexin (Fig. 1A). This recorded: locomotion score (number of squares crossed on the floor twofold increase in the total levels of ␣-internexin had no effect grid), frequencies (number of times in a test period) of rearing and on the expression levels of the NFTPs in the transgenic mice (Fig. defecation, and duration (as a percentage of the test period) of grooming. 1A). Immunoblotting and immunocytochemical analyses per- The rotorod test was used to measure motor coordination and balance (Janicke et al., 1983; Luo et al., 1996). The rotorod apparatus, Rotarod formed on various tissues such as brain, spinal cord, heart, lung, MECO, was purchased from Columbus Instruments and consists of a liver, spleen, kidney, stomach, intestines, and muscle showed that 1.75 inch rod with a rubber outer texture for grip. The rotorod speed was expression of rat ␣-internexin in the transgenic mice was re- electronically gauged. The mice were habituated on the rotorod appara- stricted to neurons. Immunocytochemical examination of the tus before testing by placing them on the rod for 1 min at the rotating ␣ speed of 2 revolutions per minute (rpm). The habituation was repeated transgenic mouse neural tissues also revealed that rat -internexin after the mice were givena1minrest. After habituation, the mice were was present in the same regions where the endogenous mouse givena5minrest and tested at a rotating speed of 10 rpm. Timing started ␣-internexin is expressed. Furthermore, the expression patterns of when the mice were placed on the rotating rod and stopped when the the transgene in the two transgenic mouse lines were found to be mice fell off from the rotating rod or after 3 min elapsed, whichever came identical. first. Aftera3minrest, the test was repeated. The retention times on the ␣ rotorod were averaged for each mouse. To increase the levels of -internexin further, we bred the To compare the performance between the 4.5-month-old hemizygous hemizygous mice within each transgenic mouse line to obtain mice and their nontransgenic littermates on the rotorod, the mice were transgenic mice that are homozygous with respect to the trans- tested at the rotating speed of 17 rpm as described above, except that gene. Western blot and densitometric analyses showed that the timing stopped when the mice fell off from the rotating rod or after 4 min ␣ elapsed, whichever came first. total -internexin levels increase to approximately threefold in Quantitations of Purkinje cells. For each transgenic mouse genotype the homozygous mice as expected (Fig. 1B), whereas the levels of examined at 12 and 18 months of age, three or four pairs of transgenic/ NFTPs remain unaltered (data not shown). nontransgenic mice per age group were used for quantitation of Purkinje cells present in cerebellum. A complete set of 10-␮m-thick paraffin- Abnormal accumulation of neuronal intermediate embedded brain sections was prepared from each mouse, and every filaments and formation of cerebellar torpedoes fifteenth section was picked and stained with cresyl violet. Comparable sections from the transgenic mice and their nontransgenic littermates Immunocytochemical analyses showed that both the hemizygous were selected for cell counting. The presence of cell nucleus was used as and homozygous mice of ␣16K-T5 and ␣16K-T21 contain abnor- a criterion for scoring the number of Purkinje cells present within each mally intense anti-␣-internexin antibody staining in the perikarya selected section. For each mouse pair, the total number of Purkinje cells from the set of the transgenic mouse sections was calculated as a per- of specific groups of neurons in the cerebellum, neocortex, and centage of that of the nontransgenic littermate. The final value of the thalamus. The abnormal immunostaining patterns, seen as early Purkinje cell number determined for each transgenic mouse genotype as 1 month of age, appeared similar between the hemizygous and group at an age point was the average of these calculated percentages homozygous mice, except that the neuronal perikarya were more obtained from quantitation of three or four pairs of transgenic/nontrans- strongly stained by the ␣-internexin antibody in the homozygotes. genic mice. Furthermore, ultrastructural studies showed that there was a greater accumulation of intermediate filaments in the neuronal RESULTS perikarya of the homozygous mice than in those of the hemizy- Neuron-specific overexpression of ␣-internexin in gous mice. Immunocytochemical and ultrastructural analyses re- transgenic mice vealed that the two mouse lines have similar morphological We previously isolated two genomic clones containing partial changes in the CNS (also see below). sequences of the rat ␣-internexin gene (Ching and Liem, 1991). In the cerebella of 4.5-month-old hemizygous and homozygous To generate transgenic mice overexpressing ␣-internexin, we mice of ␣16K-T5 and ␣16K-T21 (Fig. 2A,B), the perikarya of used the two genomic clones to construct a transgene, which Purkinje cells were heavily stained by the anti-␣-internexin anti- consists of the entire rat ␣-internexin gene with 1.2 kb of 5Ј bodies mAb␣12, which specifically recognizes rat ␣-internexin, flanking sequence and 3.5 kb of 3Ј flanking sequence. The result- and mAb␣3G10, which reacts with both rat and mouse ing 16.5 kb transgene fragment was microinjected into the pro- ␣-internexin. Immunostaining of the cerebellar white matter in- nuclei of fertilized mouse eggs. Two transgenic mouse lines, dicated colocalization of rat and mouse ␣-internexin within the ␣16K-T5 and ␣16K-T21, were established. These two lines ex- nerve fibers. Numerous fusiform swellings of the proximal por- pressed and transmitted the transgene in a Mendelian manner. To tions of Purkinje cell axons labeled by the antibodies to distinguish the expression of the transgenic product, rat ␣-internexin (Fig. 2A,B) were found in the granular layer. These ␣-internexin, from that of the endogenous mouse protein, we fusiform swellings, called torpedoes, are usually seen in neurode- prepared a monoclonal antibody that is specific for the rat pro- generative diseases involving the cerebellum and in normal aging tein. Rat and mouse ␣-internexin share high homology in their (Duchen, 1984; Hirano, 1988). These torpedoes were also labeled primary sequence but differ in a short stretch of amino acids at by an antibody to calbindin, which serves as a protein marker for their C-terminus (Chien and Liem, 1994). We therefore used a Purkinje cells (data not shown). The torpedoes were observed in bacterially produced peptide consisting of the C-terminal region the hemizygous and homozygous mice at as early as 1 month of of rat ␣-internexin as an immunogen and generated a number of age. In the nontransgenic littermates, Purkinje cell perikarya were monoclonal antibodies. One of these monoclonal antibodies, not stained by the anti-␣-internexin antibody, and no torpedoes mAb␣12, recognizes rat but not mouse ␣-internexin. Another were observed (Fig. 2C). The torpedoes and perikarya of Pur- monoclonal antibody, mAb␣3G10, recognizes both rat and mouse kinje cells of the transgenic mice were also stained by the anti- ␣-internexin (see Figs. 1 and 2 for antibody specificity). bodies to the NFTPs (Fig. 5E,F). This result is consistent with Western blot and densitometric analyses showed that wild-type the ability of ␣-internexin to coassemble with the NFTPs into Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deficits J. Neurosci., April 15, 1999, 19(8):2974–2986 2977

Figure 1. Western blot analysis of ␣-internexin and NFTPs in transgenic mice. A, Total cellular proteins extracted from the brains (with cerebella removed) (Br), cerebella (Cb), and spinal cords (S)of 3-month-old ␣16K-T5 and ␣16K-T21 hemizygous mice and nontransgenic (NT) littermates were separated by SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose ﬁlters. The ﬁlters were immunostained with anti-␣-internexin antibodies mAb␣12 (a)ormAb␣3G10 (b); monoclonal antibodies to NF-L (c), NF-M (d), or NF-H (e). Several different exposures of the autoradio- grams were used for densitometric analysis: rat and mouse ␣-internexin were expressed at approximately the same levels, except that the level of rat ␣-internexin in the Br fraction of ␣16K-T5 is ϳ1.2 times that of mouse ␣-internexin; the endogenous levels of NFTPs remain unaltered. B, Western blots containing total cellular proteins extracted from the brains (with cerebella removed) (Br), cerebella (Cb), and spinal cords (S) of 3-month-old ␣16K-T5 and ␣16K-T21 homozygous mice and nontransgenic (NT) littermates were immunostained with anti-␣- internexin antibody mAb␣3G10. Densitometric analysis showed that rat ␣-internexin was expressed at ϳ2 times the level of mouse ␣-internexin in the homozygotes, except that the level of rat ␣-internexin is ϳ2.5 times that of mouse ␣-internexin in the Br fraction of ␣16K-T5.

filaments (Ching and Liem, 1993, 1998) and the colocalization of Immunocytochemical examination of the neocortex of 4.5- ␣-internexin with the NFTPs in the CNS axons (Kaplan et al., month-old hemizygous and homozygous mice of the two trans- 1990). Thus, the immunocytochemical data showed that overex- genic lines also showed the presence of filamentous inclusions in pression of ␣-internexin in the transgenic mice caused the forma- the perikarya of large pyramidal neurons in layers III and V that tion of torpedoes and abnormal accumulation of neuronal inter- were intensely stained by the ␣-internexin antibodies, mAb␣12 ␣ mediate filaments (composed of -internexin and NFTPs) in the and mAb␣3G10 (Fig. 5A), as well as by the antibodies to the perikarya of Purkinje cells. NFTPs (Fig. 5C). Although these filamentous inclusions have a Ultrastructural analysis of the abnormal Purkinje cells by elec- neurofibrillary tangle-like appearance, they were not stained by tron microscopy revealed a swirl of closely packed, randomly an anti-tau antibody that recognizes both the phosphorylated and oriented neuronal intermediate filaments in the cytoplasm (Fig. nonphosphorylated forms of tau (data not shown). Because tau is A 3 ). The nucleus was eccentrically localized, and many cellular the major constituent of the paired helical filaments (Kondo et al., organelles were pushed to the periphery by the masses of neuro- 1988; Goedert et al., 1989; Brion et al., 1991), the absence of tau nal intermediate filaments (data not shown). Some of the mito- immunostaining indicated that the filamentous inclusions in the chondria that were trapped within the neurofilamentous masses perikarya of these neurons differ from the neurofibrillary tangles looked abnormal or smaller than normal and often associated of Alzheimer’s disease. Similar perikaryal staining was also de- with the smooth endoplasmic reticulum (Fig. 3A). The bulk of the torpedo consists of masses of closely packed, randomly oriented tected in the ventral anterior nucleus and ventral posteromedial neuronal intermediate filaments and aggregates of organelles nucleus of the transgenic mouse thalamus (Fig. 5B). Examination (Fig. 4A,B). Some mitochondria and smooth endoplasmic retic- of these abnormal neurons in the neocortex and thalamus by ulum looked abnormal, and their membranes appeared to be electron microscopy revealed the presence of massive levels of continuous with each other. These features are typical of torpe- closely packed, randomly oriented neuronal intermediate fila- does found in human neurodegenerative diseases (Mann et al., ments in the cytoplasm and an eccentrically displaced nucleus 1980). The Purkinje cell axon distal to the Purkinje cell soma and (Fig. 3B). Cellular organelles such as some of the mitochondria torpedo contains high levels of neuronal intermediate filaments and smooth endoplasmic reticulum that were trapped within the (Fig. 4C). Despite the perikaryal accumulation of neuronal inter- neuronal intermediate filaments looked abnormal and often ag- mediate filaments and the formation of torpedoes, no significant gregated, but those that were squeezed to the periphery by the loss of neurons was observed at 4.5 months of age. neuronal intermediate filaments appeared normal (Fig. 3B). In 2978 J. Neurosci., April 15, 1999, 19(8):2974–2986 Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deficits

Figure 2. Indirect immunofluorescence staining of cerebella from 4.5-month-old transgenic and nontransgenic mice with anti-␣-internexin antibodies. Sagittal sections (15 ␮m) of cerebella from ␣16k-T5 (A) and ␣16k-T21 (B) hemizygous mice and nontransgenic littermates (C, D) were stained with anti-␣-internexin antibodies. A and C were stained with mAb␣3G10, which detects both mouse and rat ␣-internexin. B and D were stained with mAb␣12, which specifically recognizes rat ␣-internexin. Perikaryal immunostaining of Purkinje cells and torpedoes were detected in the transgenic mice, but not in their nontransgenic littermates. p, Purkinje cell; t, torpedo; m, molecular layer; g, granular layer. Scale bar, 45 ␮m. contrast to the Purkinje cells, prominent swellings of proximal antibodies, SMI31, SMI32, and SMI36, were used for immuno- axons were not seen in these neurons although higher neurofila- cytochemical staining. SMI31 and SMI36 detect phosphorylated ment density was found in the axons. Furthermore, no obvious epitopes, whereas SMI32 recognizes only nonphosphorylated neuronal loss was observed. forms of NF-M and NF-H. The results showed that the perikarya Abnormal phosphorylation of NF-M and NF-H in perikaryal of the neurons containing neurofilamentous inclusions were neurofilamentous inclusions is often a hallmark of human neuro- stained by SMI32, but not by SMI31 and SMI36, indicating that degenerative diseases (Schmidt et al., 1987; Kato and Hirano, the NF-M and NF-H accumulated in the neuronal perikarya were 1990; Nakazato et al., 1990; Sobue et al., 1990). To determine not phosphorylated (Fig. 5C–F). In contrast, the cerebellar tor- whether NF-H and NF-M in the perikarya of the cerebellar pedoes were stained by all three antibodies, demonstrating the Purkinje cells, neocortical pyramidal neurons, and thalamic neu- presence of both phosphorylated and nonphosphorylated forms rons described above were phosphorylated, three monoclonal of NF-M and NF-H in the torpedoes (Fig. 5E,F). Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deficits J. Neurosci., April 15, 1999, 19(8):2974–2986 2979

Figure 3. Ultrastructural analysis of Purkinje cells and neocortical pyramidal neurons from 4.5-month-old transgenic mice. A, Ultrathin sections of cerebellum from an ␣16k-T5 homozygous mouse examined by electron microscopy revealed accumulation of high levels of intermediate filaments in the soma of a Purkinje cell. Lying within the neurofilamentous masses are aggregates of mitochondria and sER, some of which appear to associate with each other (arrows). Although the external membranes of these mitochondria appear to be continuous with sER, some other mitochondria look normal. B, Electron micrograph showed massive accumulation of intermediate filaments in the soma of a neocortical pyramidal neuron from a 4.5-month-old ␣16k-T5 homozygous mouse. Note that the nucleus is eccentrically localized, and many cellular organelles such as mitochondria are peripherally displaced. Some mitochondria trapped within the masses of neuronal intermediate filaments often aggregate and appear abnormal. The neuronal intermediate filaments in A and B are highly packed and randomly oriented. m, Mitochondria; sER, smooth endoplasmic reticulum; n, nucleus. Scale bars: A, 0.75 ␮m; B, 2.0 ␮m.

Examination of the spinal cords of the transgenic mice did not Deficit in motor coordination of transgenic mice reveal any perikaryal accumulation of neuronal intermediate filaments in motor neurons or axonal swellings, although expression Neither the hemizygous nor homozygous mice of the ␣16K-T5 of the transgene increases the levels of ␣-internexin in the spinal and ␣16K-T21 lines showed tremors or any other overt symptoms cord to ϳ3 times the normal levels in the homozygotes. Consis- of disorders. They also appeared to reproduce normally, similar tent with the normal morphology observed in the spinal cord, no to their nontransgenic littermates. To assess any possible deficits skeletal muscle atrophy was found in the transgenic mice (data in motor performance of the transgenic mice resulting from the not shown). observed neuronal pathology, we subjected the mice to a battery 2980 J. Neurosci., April 15, 1999, 19(8):2974–2986 Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deficits

motor performances in preliminary behavioral tests, they were pooled into the same groups for behavioral analysis. Analysis by the open-field test revealed no significant differences ( p Ͼ 0.05, F test) in locomotion, grooming, and rearing between the transgenic and nontransgenic mice (Table 1). Al- though the frequency of defecation was found significantly smaller in the 3-month-old homozygous transgenic mice ( p Ͻ 0.05) compared with that of the nontransgenic mice, it was not significant ( p Ͼ 0.05) among the 6-month-old mice. Taken together, these results indicate that the overall locomotor activity and gross motor-posture patterns of the transgenic mice were indistinguishable from those of the nontransgenic mice. The mice were subsequently tested for motor coordination and balance on a rotorod. The results showed significant differences ( p Ͻ 0.001, F test) in the retention times on the rotorod between genotype groups (Fig. 6). The 3- and 6-month-old homozygous mice performed more poorly than the age-matched hemizygous and nontransgenic groups. There were no significant differences ( p Ͼ 0.05) in performance between the hemizygous and nontransgenic mice at a rotating speed of 10 rpm (Fig. 6A). To determine whether the ␣-internexin-overexpressing hemizygous mice show impairment in motor coordination on a rotorod at a higher speed, new groups of 4.5-month-old hemizygous and nontransgenic mice were tested at a rotating speed of 17 rpm. At this speed, the retention times of the hemizygous mice were much shorter than those of the age-matched nontransgenic mice (Fig. 6B). These results suggest that both the hemizygous and homozygous mice of ␣16K-T5 and ␣16K-T21 are impaired in the ability to perform a complex pattern of movements involving high de- mands in motor coordination and balance. Furthermore, the poorer performance of the homozygous mice, compared with the hemizygous mice, indicates that the motor impairment is transgene dosage-dependent.

Neuronal degeneration in transgenic mice Examination of the brains from the older hemizygous and homozygous mice (12–18 months of age) of ␣16K-T5 and ␣16K-T21 at the light and electron microscopic levels revealed neuronal degeneration that is progressive with increasing age of the mice (Figs. 7, 8, Table 2). Loss of neurons was seen in the neocortex, thalamus, and cerebellum of aged transgenic mice (Fig. 7). Ultra- structural analysis of the brains from 13-month-old transgenic mice showed degenerating and degenerated processes in the molecular layer (Fig. 8A) as well as degenerating torpedoes and degenerated axons in the granular layer (Fig. 8B). Degenerated and degenerating axons and processes were also found in the neocortex (Fig. 8C) and the thalamus (data not shown). In all Figure 4. Ultrastructural analysis of cerebellar torpedo in transgenic mice. A, Electron micrograph of the cerebellum from a 4.5-month-old three brain regions, microglia were often detected at the neuronal ␣16k-T21 homozygous mouse showed the presence of a torpedo (arrow) degeneration sites (Fig. 8C). High levels of neuronal intermediate in the granular layer. B, High-magnification micrograph of the torpedo (A, filaments were still present in the perikarya of Purkinje cells, squared area) revealed the presence of closely packed, randomly oriented neocortical pyramidal neurons, and thalamic neurons. In contrast, neuronal intermediate filaments and aggregates of organelles. C, High- degenerated processes and axons were very rarely found in the magnification micrograph of the Purkinje cell axon (A, squared area) distal to the torpedo revealed the presence of high levels of neuronal nontransgenic littermates. These data showed that misaccumula- intermediate filaments. Scale bars: A, 4.0 ␮m; B, 0.4 ␮m; C, 0.5 ␮m. tion of high levels of neuronal intermediate filaments ultimately led to neuronal degeneration in the older transgenic mice. Examination of the cerebella from the hemizygous and ho- of behavioral tests. Two age groups (3 and 6 months) of homozy- mozygous transgenic mice at 12–18 months of age revealed pro- gous, hemizygous, and nontransgenic mice were tested, and their gressive loss of Purkinje cells (Fig. 7A–D). Quantitative analysis performances were compared. The mice within each age group of Purkinje cell numbers showed that at 12 months of age, the were weight-matched. Because the two transgenic mouse lines hemizygous and homozygous mice had a decrease of 16.6 and showed identical expression patterns, neuronal pathologies and 37.4%, respectively, in the Purkinje cell population (Table 2). By Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deficits J. Neurosci., April 15, 1999, 19(8):2974–2986 2981

Figure 5. Immunocytochemical analysis of brains from 4.5-month-old transgenic mice. Sagittal sections of neocortex (A, C, D), thalamus (B), and cerebellum (E, F) from ␣16k-T5 homozygous mice were stained with antibodies mAb␣3G10, which reacts with both rat and mouse ␣-internexin (A, B), SMI32, which detects only the nonphosphorylated forms of NF-M and NF-H (C, E), or SMI36, which recognizes phosphorylated epitopes on NF-M and NF-H (D, F). Perikaryal immunostainings were observed with mAb␣3G10 and SMI32, but not with SMI36. In contrast, torpedoes were stained by both SMI32 and SMI36. p, Purkinje cell; t, torpedo. Scale bar, 35 ␮m.

18 months of age, there is a further decrease in the number of zygous mice. Taken together, these data demonstrated that mis- Purkinje cells, with a 60.6 and 71.5% loss in the hemizygous and accumulation of high levels of neuronal intermediate ﬁlaments in homozygous mice, respectively. Overall, the extent of Purkinje Purkinje cells ultimately led to the degeneration and death of cell loss was greater in the homozygous mice than in the hemi- these neurons. 2982 J. Neurosci., April 15, 1999, 19(8):2974–2986 Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deﬁcits

Table 1. Motor and posture patterns of transgenic and nontransgenic mice measured in open-ﬁeld test

Mice 3 months of age 6 months of age Nontransgenic Hemizygous Homozygous Nontransgenic Hemizygous Homozygous Locomotion score 333 Ϯ 121 331 Ϯ 99 317 Ϯ 102 217 Ϯ 90 271 Ϯ 134 236 Ϯ 101 Rearing frequency 64.7 Ϯ 28.5 57.1 Ϯ 24.5 62.1 Ϯ 21.2 37.8 Ϯ 15.2 40.3 Ϯ 24.0 43.4 Ϯ 25.2 Grooming time (%) 5.62 Ϯ 3.65 4.78 Ϯ 3.25 5.38 Ϯ 4.11 7.28 Ϯ 5.34 7.67 Ϯ 5.29 5.30 Ϯ 3.79 Defecation frequency 5.6 Ϯ 1.6 4.9 Ϯ 1.7 4.1 Ϯ 1.2* 5.4 Ϯ 2.2 4.2 Ϯ 2.5 5.5 Ϯ 2.4

Locomotion score was measured as the number of squares crossed, frequency as the number of times in the test period, and grooming time as a percentage of the test period. Values given are means Ϯ SD. *Denotes statistical signiﬁcance ( p Ͻ 0.05), found only in one parameter: defecation frequency of the 3-month-old homozygous mice is signiﬁcantly different from that of the nontransgenic mice of the same age.

mice and as a result, ␣-internexin as well as the NFTPs accumu- late in the neuronal perikarya and swollen axons. Unlike some of the transgenic mice that express wild-type or mutant NFTPs (Cote et al., 1993; Xu et al., 1993; Eyer and Peterson, 1994; Lee et al., 1994; Wong et al., 1995; Marszalek et al., 1996), the ␣16K-T5 and ␣16K-T21 transgenic mice did not show any axonal swellings and perikaryal accumulation of neuronal intermediate filaments in the motor neurons of the spinal cord, although the expression levels of ␣-internexin increased in these neurons. Consistent with this result, they also did not develop any skeletal muscle atrophy or ALS-like phenotype. Because ␣-internexin is normally expressed at much lower levels than the NFTPs in the motor neurons of the spinal cord (Chiu et al., 1989; Kaplan et al., 1990; Fliegner et al., 1994), the increased levels of ␣-internexin may still be insufficient to cause misaccumulation of neuronal intermediate filaments in motor neurons. Figure 6. Comparison of performance in rotorod test between trans- In contrast, morphological analyses of the brains of the genic and nontransgenic mice. A, ␣-Internexin-overexpressing hemizy- ␣16K-T5 and ␣16K-T21 mice revealed different groups of neu- gous (HZ) and homozygous (HOM) mice of 3 and 6 months of age and rons that are susceptible to the overexpression of ␣-internexin. their age-matched nontransgenic (NT) littermates (n ϭ 10 each group) were tested, and their retention times (in seconds) on the rotorod at a Abnormal perikaryal accumulations of neuronal intermediate speed of 10 rpm were compared. The ␣-internexin-overexpressing ho- filaments were detected in the Purkinje cells of the cerebellum, mozygous mice, but not the hemizygous mice, performed poorly com- the large pyramidal neurons in layers III and V of the neocortex, ϭ pared with their nontransgenic littermates (3-month-old mice, F(2,27) Ͻ ϭ Ͻ and the ventral anterior nucleus and ventral posteromedial nu- 56.7, p 0.001; 6-month-old mice, F(2,27) 27.5, p 0.001. B, ␣-Internexin-overexpressing hemizygous (HZ) mice of 4.5 months of age cleus of the thalamus. Prominent swellings of the Purkinje cell and their age-matched nontransgenic (NT) littermates (n ϭ 15 each axons also occurred, resulting in the presence of numerous tor- group) were tested at a speed of 17 rpm on the rotorod, and their pedoes in the granular layer of the cerebellum. Thus, Purkinje retention times (in seconds) were compared. There is a significant differ- cells appear to be the most affected by the overexpression of Ͻ ence ( p 0.001; F test) in the retention times between the hemizygous ␣-internexin. Although these abnormal morphological changes mice and their nontransgenic littermates. were detected as early as 1 month of age, prominent loss of the affected neurons was seen mainly in the aged (Ն12 months) DISCUSSION transgenic mice, indicating a remarkable degree of tolerance of Our studies demonstrate that overexpression of ␣-internexin in- the neurons for massive misaccumulation of neuronal intermedi- duces the formation of cerebellar torpedoes and abnormal accu- ate filaments. This neuronal tolerance may contribute in part to mulations of neuronal intermediate filaments (composed of the slow process of neuronal degeneration and hence the gradual ␣-internexin and the NFTPs) in the brains of the ␣16K-T5 and progression of neurodegenerative diseases with neurofilamentous ␣16K-T21 hemizygous and homozygous mice as early as 1 month inclusions. of age. Furthermore, these mice exhibit a transgene dosage- Consistent with the slow process of neuronal degeneration in dependent deficit in motor coordination as early as 3 months of neurodegenerative diseases, the neurodegeneration and ultimate age. Because there is no obvious loss of neurons at this age, the loss of neurons observed in the old ␣-internexin-overexpressing observation of a deficit in the motor coordination of the trans- transgenic mice (12–18 months of age) are progressive with genic mice indicates that the functions of the affected neurons are increasing mouse age. By 18 months of age, prominent loss of compromised because of misaccumulations of high levels of neu- Purkinje cells (Ն60%) was observed in both hemizygous and ronal intermediate filaments. The mechanism by which the accu- homozygous mice. Furthermore, the extent of neuron loss is mulations of neuronal intermediate filaments occur in these ani- proportional to the levels of misaccumulated neuronal interme- mals is not clear. It is possible that the system responsible for diate filaments, as indicated by the greater loss of Purkinje cells in transporting neuronal intermediate filaments to the axons is over- the homozygous mice compared with the hemizygous mice. Thus, loaded by excessive amounts of ␣-internexin in the transgenic our data yield direct evidence that misaccumulation of high levels Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deficits J. Neurosci., April 15, 1999, 19(8):2974–2986 2983

Figure 7. Neuronal degeneration in aged transgenic mice. Sagittal paraffin-embedded sections (10 ␮m) were stained with cresyl violet and examined by light microscopy. A–D, Examination of the cerebella from 12-month-old nontransgenic (A) and homozygous (B) mice, and 18-month-old nontransgenic (C) and homozygous (D) mice revealed progressive loss of Purkinje cells as the transgenic mice aged (C, D). Arrowhead in B shows a Purkinje cell containing neurofilamentous inclusion. m, Molecular layer; p, Purkinje cell; g, granular layers. E, F, Examination of the thalamus from 18-month-old nontransgenic (E) and homozygous mice (F) showed the presence of degenerated neurons (arrow) and neurons containing neurofilamentous inclusions (arrowhead) in the transgenic mice. Scale bars: A–D,50␮m; E, F,50␮m. of neuronal intermediate filaments arising from overexpression of neuronal intermediate filaments, as shown by the poorer perfor- ␣-internexin can cause progressive neurodegeneration and neu- mance of the homozygous mice, compared with the hemizygous ron death. mice, in the rotorod test. Moreover, this neuronal dysfunction The transgene dosage-dependent motor coordination deficits occurs before the neuronal degeneration and loss seen in the observed in the ␣-internexin-overexpressing transgenic mice transgenic mice. The resulting deficit in the motor coordination of demonstrate that high levels of misaccumulated neuronal inter- the transgenic mice is consistent with the observed morphological mediate filaments can compromise the normal functions of neu- neuronal changes. Motor coordination requires the normal oper- rons containing filamentous inclusions. The degree of this neu- ation of a number of neural structures, which also include the ronal dysfunction is proportional to the levels of misaccumulated motor cortex, thalamus, and cerebellum (Thach et al., 1992; 2984 J. Neurosci., April 15, 1999, 19(8):2974–2986 Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deficits

Table 2. Quantitation of Purkinje cell numbers in transgenic mice overexpressing ␣-internexin

Genotype Age (months) Number of Purkinje cells present (%)* Hemizygous 12 83.4 Ϯ 2.1 18 39.4 Ϯ 4.4 Homozygous 12 62.6 Ϯ 3.7 18 28.5 Ϯ 5.1

*Numbers of Purkinje cells present in the transgenic mice are expressed as percentages of those in the nontransgenic littermates. Values given are means Ϯ SD (from quantitation of three or four pairs of transgenic/ nontransgenic mice in each group).

Llinas and Welsh, 1993; Ivry, 1997). These neural structures together control and regulate movement specification and initia- tion. The cerebellum, in particular, is widely believed to play a crucial role in coordinating and modulating unskilled and skilled movements and is implicated in regulating the temporal patterns of movement. It also directs its output to the cerebral cortex and thalamus. Purkinje cells, the neurons most affected by the overexpression of ␣-internexin, send their axons to the deep cerebellar nuclei, the major output pathway from the cerebellum. The abnormal pyramidal neurons containing neurofilamentous inclusions are located in the premotor and motor areas of the neocortex. Thus, it is not surprising that the transgenic mice with the morphological neuronal changes in the cerebellum, neocortex, and thalamus are impaired in motor coordination. Misaccumulation of high levels of neuronal intermediate filaments may induce neuronal dysfunction and degeneration by at least two mechanisms. The findings of some abnormal cellular organelles (such as the mitochondria and smooth endoplasmic reticulum) entrapped within the perikaryal neurofilamentous inclusions and an increase in the number of these abnormal organelles in the older ␣-internexin-overexpressing transgenic mice suggest that the cellular metabolism may be perturbed because of the organelle damages and a shortage of normal organelles re- quired for neuronal function and viability, ultimately resulting in neuronal dysfunction and degeneration. Moreover, the neuronal intermediate filaments present in the cerebellar torpedoes and perikarya of the affected neurons in the ␣-internexin-overexpressing transgenic mice are randomly oriented. Excessive accumulation of these disorganized neurofilamentous cytoskeletons may cause defects in axonal transport of neurofilaments and other cellular components essential for axonal maintenance. It has previously been shown that overexpression of human NF-H in transgenic mice impairs the axonal transport of NFTPs and cellular organelles (Collard et al., 1995). Retardation in the slow axonal transport of cytoskeletal elements during aging (McQuar- rie et al., 1989) may further compound the impairment of axonal transport caused by the abnormal accumulation of neuronal intermediate filaments, thereby accelerating axonal degeneration in older transgenic mice. The presence of phosphorylated NF-M and NF-H in perikaryal neurofilamentous inclusions is often a pathological Figure 8. Ultrastructural analysis of the cerebellum and neocortex from hallmark of many neurodegenerative diseases (Schmidt et al., 13-month-old transgenic mice. Electron micrographs of the cerebellum 1987; Kato and Hirano, 1990; Nakazato et al., 1990; Sobue et al., ␣ (A, B) and neocortex (C) from 13-month-old 16k-T5 homozygous mice 1990). This abnormal presence of phosphorylated NF-M and revealed degenerated processes (arrows) in the molecular layer (A) and a degenerating torpedo (arrow) in the granular layer (B) of the cerebellum, NF-H in neuronal perikarya has also been observed in some of and degenerated processes (arrows) and nearby microglial cells in the the transgenic mice overexpressing wild-type or mutant NFTPs neocortex (C). Scale bars: A, 2.0 ␮m; B, 1.5 ␮m; C, 1.5 ␮m. (Xu et al., 1993; Eyer and Peterson, 1994; Lee et al., 1994; Tu et al., 1997). Study of neurofilamentous aggregates induced in cul- tured neurons suggests that perikaryal accumulation of neuronal Ching et al. • ␣-Internexin-Overexpressing Mice with Motor Deficits J. 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